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Biomedical subjects

J E Manning

Publications and source records attributed to J E Manning.

At least 55 records · Page 3Linked to original sources
PubMed1991

Nucleotide sequence and transcription of a trypomastigote surface antigen gene of Trypanosoma cruzi.

In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes.

Amino Acid Sequence↗
PubMed1990

Trypanosoma cruzi: identification of a surface antigen restricted to the flagellar region of the infective form of the parasite.

A hybridoma cell line was derived from spleen cells of B6D2 mice infected with the Peru strain of Trypanosoma cruzi. The monoclonal antibody produced by this hybridoma, designated mAb20H1, reacts exclusively with molecular components of trypomastigotes, the infective form of the parasite. The results of indirect immunofluorescence and of immunoelectron microscopy with gold-tagged antibodies indicate that the 20H1 antigen is restricted to the surface of the part of the flagellum in contact with the cell body and to the surface of the cell body in the immediate vicinity of this organelle. Western blot analysis showed that the 20H1 antigen consists of four to five different molecules with sizes between 34 and 41 kDa, and that these molecules are glycoproteins with affinity for concanavalin A. In other strains of T. cruzi, mAb20H1 reacts with glycoproteins with apparent sizes that range between 37 and 43 kDa in the CL, Esmeraldo and Y strains, and between 41 and 45 kDa in the Silvio strain.

Animals↗
PubMed1989

The 85-kd surface antigen gene of Trypanosoma cruzi is telomeric and a member of a multigene family.

In previous studies we identified a 500 bp DNA fragment from the genome of Trypanosoma cruzi which encoded epitopes present in an 85 kd trypomastigote-specific surface antigen. A unique feature of this DNA insert was the presence of a 27 bp tandem repeat unit within the putative coding region. The findings presented here show that the gene which encodes this particular surface protein is a member of a multigene family, and that the 27 bp repeat unit defines a subset of this family. Only four separate members of the family contain sequences homologous to the 27 bp repeat unit. Of these, three have been cloned and shown by direct nucleotide sequence analysis not to contain the original 500 bp fragment. By restriction enzyme analysis, the 500 bp fragment is inferred to be present in a 5.4 kb EcoRI genomic DNA fragment that is refractory to isolation by standard cloning procedures. Preferential sensitivity of this fragment to digestion with Bal31 nuclease indicates that it is likely to be telomeric, thus explaining the inability to obtain it in several different recombinant DNA libraries. In order to determine which of the four members were transcribed, 26 cDNA recombinants having sequence homology with the 27 bp repeat were examined. Restriction enzyme maps and nucleotide sequence analysis of these cDNAs indicate that transcription occurs almost exclusively from the telomeric member of the family.

Animals↗
PubMed1989

Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster.

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.

Animals↗
PubMed1989

The LSP1-alpha gene of Drosophila melanogaster exhibits dosage compensation when it is relocated to a different site on the X chromosome.

The LSP1-alpha gene of Drosophila melanogaster is located on the X chromosome at 11B yet is not dosage compensated. In order to determine if this gene is inherently incapable of dosage compensation or if it does not compensate because the appropriate regulatory cis-acting sequences are absent from its chromosomal domain, we have undertaken to relocate it to ectopic sites on the X chromosome. To differentiate between the transcripts produced by the transduced gene and those produced by the indigenous gene, we inserted a 500-bp sequence of mouse DNA into the LSP1-alpha clone prior to using it for transformation. Our results show that the LSP1-alpha gene exhibits equivalent levels of transcripts in the two sexes when it is relocated to either an autosomal site or to an ectopic site on the X chromosome. We conclude that the LSP1-alpha gene is capable of dosage compensation.

Animals↗
PubMed1989

Determination of viridans streptococci surface lipoteichoic acid by enzyme linked immune sorbent assay.

An enzyme linked immune sorbent assay (ELISA) was developed to measure bacterial surface lipoteichoic acid (LTA). Numerous strains of oral streptococci belonging to the 'viridans' group were examined on three separate occasions. The results show that, under these cultural conditions, oral streptococci do not normally express LTA on the cell surface. Occasionally strains produced amounts of LTA detectable using the ELISA but this was not a reproducible phenomenon.

Bacterial Adhesion↗
PubMed1988

Nucleotide sequence of the Drosophila glucose-6-phosphate dehydrogenase gene and comparison with the homologous human gene.

Glucose-6-phosphate dehydrogenase (G6PD) has a major role in NADPH production and is found in almost all cell types. The structural gene for G6PD is X-linked in Drosophila melanogaster, as it is in most eukaryotic organisms, and due to its ubiquitous expression, it can be considered a typical 'housekeeping' gene. Here we present the complete nucleotide (nt) sequence of G6PD cDNAs as well as the genomic copy of the G6PD gene. The G6PD gene has three introns so that the protein-coding region is divided into four segments. The 5'-end of mature G6PD mRNA is located 289 +/- 1 nt upstream from the start codon. The sequence upstream from the transcription start point is G + T-rich and contains no commonly found transcription regulatory elements, such as a TATA box or GGGCGG sequence. D. melanogaster G6PD is 65% homologous with the human G6PD protein but has no homology with the human sequence for the first 42 amino acid residues. The G6PD gene was shown to be active when transduced to autosomal positions. For each transformant, G6PD activity in both male and female adults was not significantly different, indicating that the transduced gene, unlike the resident G6PD, is not dosage-compensated in males.

Amino Acid Sequence↗
PubMed1988

Stage and strain specific expression of the tandemly repeated 90 kDa surface antigen gene family in Trypanosoma cruzi.

A recombinant cDNA library constructed in the expression vector lambda gtll using mRNA from the trypomastigote stage of Trypanosoma cruzi was screened with two monoclonal antibodies that have been shown to react with a 105 kDa and a 90 kDa surface antigen in trypomastigotes of the Peru and Y strains of T. cruzi. One recombinant lambda phage, designated Tcc-20, was reactive to both monoclonals. The beta-galactosidase/T. cruzi hybrid protein encoded in Tcc-20 is recognized by the monoclonal antibodies and by serum antibodies from mice infected with strains of T. cruzi which contain the 90 kDa antigen. Antibodies immunoselected from serum of mice infected with the Peru strain by adsorption to Tcc-20 fusion protein react specifically with a 90 kDa polypeptide in trypomastigote but not epimastigote lysates of T. cruzi. The mRNA complementary to the DNA insert in Tcc-20 is present only in those stages and strains of T. cruzi which express the 90 kDa surface antigen. These characteristics are strong evidence that the T. cruzi DNA fragment cloned into Tcc-20 encodes a portion of the 90 kDa surface antigen. The gene(s) which encodes this polypeptide is shown to be present in approximately 20 copies per haploid genome and most, and possibly all, of the copies are found in a tandemly linked multigene family.

Animals↗
PubMed1988

Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid.

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.

Bacterial Adhesion↗
PubMed1987

Gene dosage compensation in Drosophila melanogaster.

The comparative lack of sexual dimorphism of apricot and other [sex-linked] genes studied is due to "sex-limitation", i.e., to a compensatory influence of the dosage difference between the sexes in respect to other genes in the X-chromosome. The facts are of particular interest from an evolutionary standpoint.

Alleles↗
PubMed1987

The hydrophobicity of 'viridans' streptococci isolated from the human mouth.

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower (P less than 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.

Bacterial Adhesion↗
PubMed1986

Variation in antigenic determinants specific to the infective stage of Trypanosoma cruzi.

Monoclonal antibodies reactive with the surface antigens of the Peru strain of Trypanosoma cruzi were analyzed by Western blots and immunofluorescence assays to determine their reactivity with three life cycle stages and five strain isolates of T. cruzi. One monoclonal antibody, 7.6, recognized a 68-kilodalton (kDa) polypeptide in Western blots of Peru strain trypomastigotes, epimastigotes, and amastigotes. A 68-kDa polypeptide was also detected by monoclonal antibody 7.6 in trypomastigotes of the CL and Y strains and in the clonal isolates Esmeraldo clone 3 and Silvio X10 clone 1. Positive immunofluorescence results were obtained for all life cycle stages of the five strains that were reacted with monoclonal antibody 7.6, thus indicating that the antigen recognized by monoclonal antibody 7.6 is universally present in all T. cruzi strains tested. In contrast, monoclonal antibody 4.2 reacted with a polypeptide doublet of 90 and 105 kDa in Western blots of Peru strain trypomastigotes, but it did not detect these antigens in epimastigotes or amastigotes. The same polypeptide doublet of 90 and 105 kDa was also detected in Western blots of Y strain trypomastigotes; however, no bands were detected in blots of strain CL or isolate Silvio X10 clone 1 trypomastigotes. In blots of Esmeraldo clone 3 trypomastigotes, a single band of 130 kDa was detected by monoclonal antibody 4.2. In immunofluorescence assays of monoclonal antibody 4.2, positive reactions were obtained only with trypomastigotes of Peru, Y, and Esmeraldo clone 3 strains. Thus, monoclonal antibody 4.2 recognizes a trypomastigote-specific antigen which is not universally present on all strains of T. cruzi.

Animals↗
PubMed1985

Identification of monoclonal antibodies against the trypomastigote stage of Trypanosoma cruzi by use of iminobiotinylated surface polypeptides.

The surface polypeptides of epimastigotes and tissue culture-derived trypomastigotes of Trypanosoma cruzi have been isolated free of most cytosolic components by use of the 2-iminobiotin-avidin interaction. Polypeptides of the trypomastigote stage obtained by this technique are recognized by serum antibodies from Chagasic patients and T. cruzi-infected mice. These polypeptides have been used as the detecting antigen for the identification of hybridoma cells producing monoclonal antibodies against the surface proteins of the trypomastigote stage of T. cruzi. These experiments document a practical approach for obtaining T. cruzi surface proteins in sufficient quantity and purity for use in immunological studies.

Animals↗
PubMed1985

Isolation and characterization of the glucose-6-phosphate dehydrogenase gene of Drosophila melanogaster.

To investigate the molecular basis of dosage compensation in Drosophila, a recombinant lambda phage containing the Drosophila melanogaster glucose-6-phosphatase dehydrogenase (G6PD) gene was isolated by differential screening of a Drosophila genomic lambda library with poly(A)+RNA obtained from polyribosomes enriched for or depleted of G6PD mRNA sequences. Of 44 000 plaques screened, a single phage, lambda DmG21, showed hybridization with the enriched poly(A)+RNA but not the depleted one. Confirmation that the Drosophila DNA fragment cloned in lambda DmG21 contains the G6PD gene sequence is based on the following observations. lambda DmG21 DNA shows hybridization only to the 18D region of the salivary gland X-chromosome, which is the known cytological locus for the G6PD gene. In vitro translation of the poly(A)+mRNA selected by hybridization to lambda DmG21 DNA sequences shows a polypeptide product of apparent Mr 55 000, identical to that of the monomeric unit of G6PD. When the putative coding sequence of G6PD is cloned into the expression vector lambda gt11, recombinant plaques are recognized by anti-G6PD immunoglobulin. A transcriptional map of the G6PD gene shows that it is divided into two exons, 0.9 kb (exon I) and 1.8 kb (exon II) long, which are separated by a 2.4-kb intron. The G6PD mRNA is 2.0 kb in length and the steady-state level of the mRNA is similar in both sexes. Measurement of the copy number of the G6PD gene in males and females shows the gene to be present once per X-chromosome in both sexes. No amplification of the gene sequence was observed in males. These results are, therefore, in agreement with the previous suggestion that dosage compensation is the result of enhanced transcription of X-linked genes in males.

Animals↗
PubMed1984

Major surface proteins and antigens on the different in vivo and in vitro forms of Trypanosoma cruzi.

The surface proteins of six stages of Trypanosoma cruzi were labeled by Iodogen-catalized surface iodination and analyzed by one and two dimensional polyacrylamide gel electrophoresis. These stages included bloodstream trypomastigotes, culture-form trypomastigotes, amastigotes, staphylomastigotes, epimastigotes, and metacyclic trypomastigotes. Antigens recognized by serum antibodies were detected by Western blotting against serum from mice hyperimmunized against bloodstream trypomastigotes. Bloodstream trypomastigotes, culture-form trypomastigotes and staphylomastigotes contained several surface proteins of molecular weight (Mr) 90 000 and isoelectric points (pI) between 5.0 and 7.5. Western blotting reveals that at least two proteins of 90 000 Mr represent the major antigens seen on bloodstream trypomastigotes, culture-form trypomastigotes, staphylomastigotes and amastigotes. However, a 90 000 Mr protein was not detected by either Western blotting or surface iodination on epimastigotes or metacyclic trypomastigotes. The major surface proteins on these latter two stages were represented by several 72 000 Mr proteins with pI values between 5.2 and 5.8. An interesting result of this survey is that a 90 000 Mr surface antigen was present on staphylomastigotes, a stage which can be grown in cell free medium.

Animals↗
PubMed1984

Role of the H-2s haplotype in survival of mice after infection with Trypanosoma cruzi.

In studies of the resistance of inbred mice to infection with Trypanosoma cruzi Peru, mouse strain B10.S was the only strain which survived the infection resulting from the inoculation of 10(3) trypomastigotes. This is the only inbred mouse strain studied to survive infection. To investigate the effect of the H-2 haplotype on survival, C57BL/10 congenic mouse strains bearing H-2S recombinant haplotypes and mouse strains A.SWSn/J and SJL/J were tested for their ability to overcome the T. cruzi infection. None of the recombinant strains tested, including B10.S(7R), B10.S(8R), B10.S(9R), and B10.HTT, survived the infection, indicating that at least two or more regions of the H-2 locus must be H-2S to ensure survival. Strains A.SWSn/J and SJL/J with the H-2S haplotype did not survive, indicating that the genetic background outside the H-2 complex also influences survival. The congenic F1 hybrid (C57BL/10 X B10.S) F1 exhibited intermediate survival levels when compared with the parental strains, indicating that H-2S survival is affected by gene dosage. The F1 hybrid strain [B10.S(7R) X B10.S(8R)]F1, which possesses the complete H-2S haplotype in the trans configuration, did not survive T. cruzi infection, suggesting that H-2S-mediated survival does not operate by trans complementation.

Animals↗
PubMed1983

Heart rates in fire fighters using light and heavy breathing equipment: similar near-maximal exertion in response to multiple work load conditions.

Intense exertion is an occupational hazard inherent to fire fighting. This study was designed to look at the exertion levels that fire fighters attain during a fire fighting exercise when using (1) no self-contained breathing apparatus (SCBA), (2) light SCBA, and (3) heavy SCBA. Exertion levels were measured as a function of the heart rate increase relative to the maximum predicted heart rate determined by a standard treadmill exercise test. Five fire fighters wore electrocardiographic monitors during a routine fire fighting exercise. Heart rates increased rapidly to 70% to 80% of maximum within the first minute and then plateaued at 90% to 100% until the attack on the fire was completed. There was no significant difference between exertion levels when using no SCBA, light SCBA, and heavy SCBA (split-plot analysis of variance, p greater than .25). These results suggest that fire fighters attain an intense level of physical activity quickly and maintain that level as long as they are actively engaged in fighting fire. These results also suggest that regardless of the weight of the SCBA, if employed, fire fighters exert themselves from 85% to 100% of their maximum and adjust their work output to maintain that near-maximal level.

Adult↗